The present invention relates to a microscope for fluorescence imaging microscopy, in particular for wide-field fluorescence microscopy, including a pulsed light source and an imaging detector. The invention also relates to a corresponding method, in particular for applying the microscope of the present invention.
Very generally, the present invention concerns fluorescence microscopy, and specifically wide-field fluorescence microscopy.
Wide-field fluorescence microscopy is of great importance in biological and medical research. While in classical fluorescence microscopy, the sample is illuminated more or less point by point, in wide-field microscopy, the sample is illuminated over a large area and imaged by an objective, so that the point spread function (PSF) of the overall system is determined only by the (detection) PSF of the objective. In contrast, in a confocal microscope, the incident light is focused onto the sample, so that the overall PSF is the product of the illumination PSF and the detection PSF.
In general, in wide-field fluorescence microscopes, the amount of excitation light scattered back from the sample into the detector beam path of the microscope is significantly higher than is possible in confocal microscopes, where spatial filtering is provided by the detection pinhole. In order to nevertheless be able to acquire images under satisfactory conditions, it is necessary to suppress the scattered/reflected light to a sufficient degree. To this end, wide-field fluorescence microscopes typically use barrier filters which suppress the corresponding excitation wavelengths. Usually, filter cubes are used which, in addition to the barrier filter (emission filter), also include an excitation filter for spectral filtering of the excitation light, and a dichroic as the main beam splitter, the main beam splitter assisting both the excitation filter and the emission filter.
However, the disadvantage here is that the invariable filter characteristics do not allow for spectral tuning of the excitation light. Moreover, in the case of multiple wavelength excitation, very special filters have to be used to transmit or reflect at the appropriate spectral locations, which further restricts flexibility and, given a multitude of possible spectral excitation and detection regions, requires a number of combinations so high that it cannot be implemented by the filter wheels commonly used in practice. In addition, due to the spectral width of the corresponding filters, the detection region must have a defined spectral distance from the excitation wavelength region, as a result of which excitation light which one would wish to detect in order to increase the sensitivity of the microscope is lost in the intermediate region.
For further details on confocal microscopes, reference may be made, for example, to DE 199 06 757 A1. There, the detection light is—usually—at least partially present in the form of a directed beam which passes through a pinhole. Accordingly, it is possible to use acousto-optical devices. In contrast, in wide-field microscopy, a real image is formed in the field optics, which means that light is incident at a plurality of angles at the same time, which, in turn, seems to make the use of acousto-optical devices unsuitable or even virtually impossible. This is attributable to the fact that acousto-optical devices require an enormously large optical opening and would therefore be extremely complex and expensive.
From U.S. Pat. No. 5,252,834, it is known per se to use gating in a microscope, but not for imaging purposes, and even less for suppressing reflected/scattered light during imaging. Rather, this patent is concerned with the imaging of point spectra for characterizing the chemical composition of samples (microspectrophotometry), and does not provide for the use of imaging detectors.